Engineering Ras super binders: A step towards understanding the RAS oncogenesis

RAS is a molecular switch that functions by alternating between an inactive GDP-bound and an active GTP-bound state and serves as a master regulator of many signaling cascades. Certain mutations in Ras are frequently found in various types of cancer, thus making this protein an attractive therapeutic target. Yet, Ras has been considered undruggable for many years and only a few studies exploit RAS network for drug development. Our strategy is to inhibit Ras/effector interactions by using a natural Ras binding domain as a scaffold and converting it into a high-affinity specific binder for only one Ras state. For the inhibitor design, we picked Nore1A, an effector with the highest affinity and slowest dissociation rate to Ras and also a known tumor suppressor gene. In this work, we used a combination of computational protein design and directed evolution to reengineer Nore1A into a super binder that targets preferentially Ras-GTP or Ras-GDP states. Using the yeast surface display platform, we were able to substantially increase Nore1A affinity to each physiological Ras state. The selected mutants were expressed and purified for the biochemical, biophysical and structural characterization. In addition, we transiently transfected A549 lung cancer cell line with selected Nore1A mutants and showed that our super binders inhibit the growth and migration of A549 cells more efficiently in comparison to wild type Nore1A. Our strategy presents an attractive way for Ras inhibition and development of new therapies against cancer.